Vinyl substituted radiohalogen conjugates for protein labeling

ABSTRACT

Vinyl radiohalogenated small molecules as shown in formulas I and II: ##STR1## wherein *X is a radiohalogen, C═C is a double bonded set of sp 2  hybridized carbon atoms, and substituents R 1 , R 2 , and Y are as defined below. R 1  and R 2  are substituents independently selected from among hydrogen; alkyl or substituted alkyl, provided that any sp 2  or sp carbon atom substituted on the alkyl is separated from C═C by at least one fully substituted sp 3  carbon atom; aryl or substituted aryl, provided that the aryl is bonded directly to C═C; heteroalkyl, provided, first, that no heteroatom of the heteroalkyl bonds directly to C═C and, second, that any sp 2  or sp hybridized carbon bonded to a heteroatom of the heteroalkyl is not bonded directly to or otherwise conjugated with C═C and, third, that where a single sp 3  carbon intervenes between C═C and an sp 2  or sp carbon bonded to a heteroatom that intervening sp 3  carbon must be fully substituted; heteroaryl, provided that a heteroatom of the heteroaryl is not bonded directly with C 50 C; mixed alkylaryl, provided first, that no heteroatom is bonded to C═C and, second that either an aryl moiety of the mixed alkylaryl is directly bonded to C═C or that any aryl moiety is separated from C═C by at least one sp 3  carbon atom and, where only one sp 3  hybridized carbon atom intervenes between C═C and an aryl moiety, that intervening sp 3  carbon must be fully substituted. Y is substituent containing any of the groups described above for R 1  and R 2 , except that Y cannot be hydrogen, and bearing a functional group suitable for binding to protein under conditions that preserve the biological activity of the protein. The compounds of formulas I and II can be coupled to  proteins such as monoclonal antibodies ot provide reagents for diagnostic and therapeutic applications. 
     Also metalated precursors of compounds I and II, as well as radiophormaceutical reagent kits containing any of the subject small molecules.

TECHNICAL FIELD

This invention relates to radiohalogenated small molecules for labelingproteins, particularly antibodies, useful for clinical diagnosis andtherapy, and to methods of introducing high specific activityradiohalogens into protein molecules.

BACKGROUND OF THE INVENTION

Radiohalogenated proteins have been the object of extensive scientificstudy and promise to be useful for a variety of clinical applications,both in vitro and in vivo. For example, radioiodinated ferritin is usedin an in vitro diagnostic determination of ferritin concentration inserum. Radioiodinated thyroid stimulating hormone is employed in asimilar assay.

Radionuclides of halogens possess properties that make them veryattractive for both diagnostic imaging and radiotherapy. For example,radioiodine as iodine-123 (T1/2=13 h, 159 keV gamma, electron capture)is nearly ideal for imaging with the current gamma cameras, andiodine-131 (T1/2=8 d, 364 keV gamma, beta particle), while producingimages of lower quality, has been demonstrated to be useful in clinicalradiotherapy of the thyroid. Similarly, bromine radionuclides such asbromine-75 (T1/2=1.6 h, positron) and bromine-76 (T1/2=16 h, positron)have properties that make them attractive for positron tomographicimaging, and bromine-77 (T1/2=2.4 d, several gammas, electron capture)has properties that make it attractive for radiotherapy. Otherradiohalogens, such as fluorine-18 (T1/2=110 min, positron) andastatine-211 (T1/2=7.2 h, alpha particle), are also attractivecandidates for radioimaging and radiotherapy.

The development of monoclonal antibodies that localize in canceroustissue due to their high specificity and affinity for antigens on tumorcell surfaces has increased the prospect of clinical applications ofradiolabeled antibodies for diagnosis and/or therapy. The highspecificity of the antibodies make them desirable candidates as carriermolecules to which specific radionuclides may be attached for deliveringradioactivity to a cancer site.

Other proteins, protein fragments, modified proteins, and peptides thattend to concentrate in an organ or diseased tissue are likewisecandidates to be radiohalogenated. For example, radiohalogenatedfibrinogen could be used to localize deep vein thrombosis by in vivoimaging. Disease-altered uptake of pituitary and other peptide hormonescould be monitored in a similar manner.

Unfortunately, there are presently no routine clinical diagnostic ortherapeutic applications of radiohalogen labeled antibodies for use invivo. Direct radiohalogen labeling of antibodies and other proteins hasproved to be difficult. Antibodies exhibit varying sensitivities toradiolabeling reaction conditions, and the oxidizing reaction conditionsnecessary for standard radiohalogenations are particularly deleterious.Direct radioiodination of proteins has become routine, but very often ameasurable reduction of biological activity of the protein results. Thestability of the attached radiolabel can also vary. For example, theloss of radioiodine from antibodies has been found to be as high as 50%in 24 hours for some labeled antibodies. Radiobrominations require evenstronger oxidizing reaction conditions than radioiodinations, andattempts to radiobrominate proteins directly have met with littlesuccess unless expensive and difficult to obtain enzymes are used asoxidants. Furthermore, direct radiohalogenation of proteins occursprimarily at tyrosyl residues, and the activated phenol ring of tyrosinecontributes to an inherent electronic instability of the resultantortho-substituted radiohalogen label. The radiohalogen label is alsosubject to steric hindrance effects and may in addition be available todeiodinase enzymes which catabolize the structurally similar thyroidhormones, e.g., thyroxine.

One approach that circumvents subjecting proteins to the harsh reactionconditions necessary for direct radiohalogenations is the use of smallmolecules that can be radiolabeled in a separate reaction vessel andsubsequently coupled to proteins under mild reaction conditions. Thisapproach is the basis of the commercially available Bolton-Hunterreagent, N-succinimidyl-3-(4-hydroxyphenyl)propionate. Moderateradiolabeling yields are thereby obtained with radioiodine (35-60%yields of labeled proteins), but the stability of the radioiodine labelsuffers from the same problems as described for the chemically similarradioiodinated tyrosyl residues. Similarly, the commercially availableWood's reagent, methyl-4-hydroxybenzimidate, can be radioiodinated priorto attachment to proteins. However, the radioiodinated product is alsoplagued with the inherent instability of the ortho-iodinated phenol.Even though these reagents do not yield as stable as radiolabel asdesirable, they have been extensively used for radioiodination becauselittle deactivation of the protein results from their use.

The phenolic ring is employed in both the Bolton-Hunter and Wood'sreagents because an activated aromatic ring is required in order tointroduce high specific activity radioiodine into these molecules. Itwould be very desirable to be able to introduce radiohalogens into othersmall molecules so that the radiolabel would be more stably attached.

Recent reports in the literature describe the use of organometallicintermediates to introduce high specific activity radiohalogens intovinyl positions on alkene moieties. For example, vinylstannanes havebeen reportedly used to radiohalogenate steroid in the 17 position(Hanson, R. N., et al., J. Nucl. Med. 23: 431-436, 1982), and to labelsugars (Goodman, M. M., et al., in Sixth International Symposium onRadiopharmaceutical Chemistry, Boston, MA, June 29-July 3, 1986, papernumber 106). Use of vinyl boronic acids in the radiohalogen labeling ofsteroids has also been reported. Kabalka, G. W., et al., Synth. Commun.11: 247-251, 1981; Kabalka, G. W., et al., Applications of Nuclear andRadiochemistry, Lambrecht, R. M., et al., Eds., Pergamon Press, Newark,N.J., Chapter 17, pp. 197-203, 1981.

SUMMARY OF THE INVENTION

This invention provides vinyl radiohalogenated small molecules that canbe bonded to proteins such as monoclonal antibodies under conditionsthat preserve the biological activity of the protein. The stability ofthe bond between the radiohalogen and the carbon carbon double bond towhich it is attached is maintained by selecting the other substituentsof the alkene so as to prevent the double bond from migrating to otherpositions in the molecule. The other substituents of the alkene are alsoselected to prevent addition to the double bond when the radiolabeledprotein conjugate is introduced into a physiological milieu. Enhancedstability of the radiolabel results. The subject radiohalogenated smallmolecules are shown in formulas I and II: ##STR2## wherein *X is aradiohalogen, C═C is a double bond set of sp² hybridized carbon atoms,and substituents R₁, R₂, and Y are as defined below.

R₁ and R₂ are substituents independently selected from among hydrogen;alkyl or substituted alkyl, provided that any sp² or sp carbon atomsubstituted on the alkyl is separated from C═C by at least one fullysubstituted sp³ carbon atom; aryl or substituted aryl, provided that thearyl is bonded directly to C═C; heteroalkyl, provided, first, that noheteroatom of the heteroalkyl bonds directly to C═C and, second, thatany sp² or sp hybridized carbon bonded to a heteroatom of theheteroalkyl is not bonded directly to or otherwise conjugated with C═Cand, third, that where a single sp³ carbon intervenes between C═C and ansp² or sp carbon bonded to a heteroatom that intervening sp³ carbon mustbe fully substituted; heteroaryl, provided that a heteroatom of theheteroaryl is not bonded directly with C═C; mixed alkylaryl, provided,first, that no heteroatom is bonded to C═C and, second, that either anaryl moiety of the mixed alkylaryl is directly bonded to C═C or that anyaryl moiety is separated from C═C by at least one sp³ carbon atom and,where only one sp³ hybridized carbon atom intervenes between C═C and anaryl moiety, that intervening sp³ carbon must be fully substituted.

Y is a substituent containing any of the groups described above for R₁and R₂, except that Y cannot be hydrogen, and bearing a functional groupsuitable for binding to protein under conditions that preserve thebiological activity of the protein. The compounds of formulas I and IIcan be coupled to proteins, protein fragments, immunological bindingpartners such as monoclonal antibodies and antibody fragments, plasmaproteins, peptides, and mixtures thereof to provide reagents fordiagnostic and therapeutic applications.

Also provided are metalated precursors of the foregoing radiohalogenatedsmall molecules, as well as radiopharmaceutical reagent kits containingthe subject radiolabeled and metalated small molecules.

DETAILED DESCRIPTION OF THE INVENTION

Vinyl halide bonds are as strong or stronger than arylhalide bonds andare stronger than alkylhalide bonds. The relative stability ofvinyl-halogen and aryl-halogen bond results from attachment of thehalogen onto an sp² hybridized carbon atom. Halogens attached to sp²hybridized carbon atoms are also much less prone to undergo nucleophilicsubstitution reactions than halogens attached to sp³ hybridized carbonatoms, particularly allylic and benzylic halogens; thus, the endogenousnucleophiles present in vivo are less likely to react with vinyl andaryl halides to release the anionic halide. Radiolabeled vinyl halidesmall molecules are therefore potential vehicles for stably attachingradiohalides onto proteins, provided that both the biological activityof the protein and the integrity of the sp² hybridized carbon atom towhich the radiohalide is bonded are maintained. These requirements areachieved by the subject radiohalogenated small molecules of formulas Iand II: ##STR3## wherein *X is a radiohalogen, C═C is a double bond setof sp² hybridized carbon atoms, and substituents R₁, R₂, and Y are asdefined below.

R₁ and R₂ are substituents independently selected from among hydrogen;alkyl or substituted alkyl, provided that any sp² or sp carbon atomsubstituted on the alkyl is separated from C═C by at least one fullysubstituted sp³ carbon atom; aryl or substituted aryl, provided, first,that the aryl is bonded directly to C═C and, second, that when the arylis substituted ortho or para relative to C═C the substituent does notdonate electrons to the aryl via resonance; heteroalkyl, provided,first, that no heteroatom of the heteroalkyl bonds directly to C═C and,second, that any sp² or sp hybridized carbon bonded to a heteroatom ofthe heteroalkyl is not bonded directly to or otherwise conjugated withC═C and, third, that where a single sp³ carbon intervenes between C═Cand an sp² or sp carbon bonded to a heteroatom that intervening sp³carbon must be fully substituted; heteroaryl, provided that a heteroatomof the heteroaryl is not bonded directly with C═C; mixed alkylaryl,provided, first, that no heteroatom is bonded to C═C and, second, thateither an aryl moiety of the mixed alkylaryl is directly bonded to C═Cor that any aryl moiety is separated from C═C by at least one sp³ carbonatom and, where only one sp³ hybridized carbon atom intervenes betweenC═C and an aryl moiety, that intervening sp³ carbon must be fullysubstituted. By fully substituted sp³ carbon atom as used herein ismeant a carbon atom not substituted with hydrogen or any substituentsuch that isomerization of C═C can take place.

Y is a substituent containing any of the groups described above for R₁and R₂, except that Y cannot be hydrogen, and bearing a functional groupsuitable for binding to protein under conditions that preserve thebiological activity of the protein. The compounds of formulas I and IIcan be coupled to proteins, protein fragments, immunological bindingpartners such as monoclonal antibodies and antibody fragments, plasmaproteins, peptides, and mixtures thereof to provide reagents fordiagnostic and therapeutic applications.

As utilized herein, the symbol "*X" indicates any radioisotope of:iodine, particularly ¹²³ I, ¹²⁵ I, and ¹³¹ I; bromine, particularly ⁷⁵Br, ⁷⁶ Br, and ⁷⁷ Br; fluorine, particularly ¹⁸ F; and, astatine,particularly ²¹¹ At. Preferred radiohalogens *X for diagnostic imagingpurposes include ¹³¹ I and most preferably ¹²³ I for imaging with gammacameras, and ¹⁸ F, ⁷⁵ Br, ⁷⁶ Br, and ¹²⁴ I for positron tomographicimaging. For clinical radiotherapy, preferred radiohalogens *X include¹³¹ I, ⁷⁷ Br, and ²¹¹ At. Preferred radiohalogens *X for in vitroradioimmunoassay purposes include ¹²⁵ I and ¹³¹ I.

Symbol "C═C" indicates a double bond set of sp² hybridized carbon atomsknown as an alkene. Each of the doubly bonded carbons has two additionalsp² orbitals available for bonding. Radiohalogen *X bonds to one ofthese sp² orbitals. Symbols "R₁ " and "R₂ " refer to two additionalvinyl substituents of the alkene, that is, to atoms or groups that arebonded to another two of the four available sp² orbitals. Substituent Ybonds to the fourth sp² orbital of C═C.

It is essential that substituents R₁ and R₂ (and also Y) do notdestabilize the radiohalogen-sp² carbon bond. For example, directlybonding a heteroatom such as oxygen or nitrogen to either of the sp²hybridized carbon atoms of C═C may change the electron character of thealkene, resulting in loss of the radiohalogen. Some heteroatoms may besuitable for bonding directly to group C═C provided that the heteroatomdoes not donate nonbonding electrons to C═C. Examples of suchheteroatoms include nitrogen when bonded to another functional groupsuch as carbonyl that will delocalize the nonbonding electrons, e.g., anitrogen in an amide bond. Thus, the substituents R₁ and R₂ may beheteroalkyl or heteroaryl groups, but is preferred that a heteroatom isnot directly bonded to group C═C. It is also particularly important thatvinyl substituents R₁ and R₂ do not render the alkene susceptible tonucleophilic addition. Examples of such nucleophilic addition to C═Cinclude additions to α, β-unsaturated carbonyl compounds in reactionsknown as Michael-type additions, as defined in March, J., AdvancedOrganic Chemistry, 3rd edition, John Wilex & Sons, New York, NY 664-666,1985, which is hereby incorporated by reference. Thus, for heteroalkylgroups, any sp² or sp carbon bonded to a heteroatom must not be bondeddirectly to or otherwise conjugated with C═C. Furthermore, where asingle sp³ hybridized carbon atom intervenes between C═C and an sp² orsp carbon that is bonded to a heteroatom, that sp³ carbon must be fullysubstituted so that an isomerization of the C═C double bond is notpossible. Otherwise, protons bonded to a sp³ carbon atom α to theheteroatom and allylic to group C═C would be acidic, leading toactivation of group C═C and migration of that double bond, which wouldproduce the highly undesirable allylic radiohalogenated small molecule.

It is also important that R₁ and R₂ be inert toward biological moleculesunder physiological conditions. For example, reducing sugars would notbe suitable R₁ and R₂ substituents, because such noninert substituentsmight lead to nonspecific glycosylation and radiolabeling of nontargetedtissues in vivo.

Permissible alkyl substituents R₁ or R₂ include protons and branched,straight-chain, or cyclic alkyl groups containing from 1 to about 12carbon atoms that are preferably substituted with protons or methylgroups. Most preferably, R₁ and R₂ are individually selected from amongprotons and methyl groups. Permissible heteroalkyl substituents R₁ or R₂include, but are not limited to, those in which the alkyl moiety eitheris substituted with substituents from the group halogen, OH, OG, O₂ CG,CO₂ G, CONH₂, CONG, CONHG, NH₂, NG₂, NHG, SH, SR, SOG, SO₂ G, SO₂ Ng₂,SO₂ NH₂, and SO₂ NHG or which contain as linking groups --O--, --NH--,--NG--, --CO--, --CO₂ --, --CONH--, --CONG--, --S--, --SO--, --SO₂ --,--CO₂ NH--, and --SO₂ NG--, wherein G is selected from alkyl of from 1to about 8 carbon atoms, alkenyl of from 1 to about 8 carbons, and aryl.

Permissible aryl substituents R₁ and R₂ include, but are not limited to,5, 6 and 7 carbon aromatic rings, with the most preferable arylsubstituent being phenyl. Permissible polynucleararyl substituents R₁and R₂ include those having up to 3 aryl rings, with each ringcontaining 5, 6, or 7 carbon atoms. Permissible heteroaryl substituentsR₁ and R₂ include, but are not limited to, 5, 6 and 7 member ringscontaining from 1 to about 3 heteroatoms selected from O, S, and N, withthe most preferable heteroaryl substituent being pyridine. The foregoingaryl, polynucleararyl, and heteroaryl groups may be substituted with asmany as 3 ionizable groups such as nitro, sulfonic acid, carboxylicacid, and amino in order to aid in the solubilization of compounds I andII in aqueous solutions. The aryl, polynucleararyl, and heteroarylsubstituents R₁ and R₂ may also be substituted with alkyl and withheteroalkyl groups containing heteroatoms such as oxygen, nitrogen, andsulfur.

Vinyl substituents R₁ and R₂ of the alkene may also be a mixed alkylarylhaving one or more sp³ carbon atoms linking group C═C to an aromaticmoiety. As described above, if there is only one intervening sp³ carbon,that carbon must be fully substituted since mild base treatment ofbenzylic protons (which are acidic) may lead to migration of the doublebond thereby destroying the radiohalogen-sp² carbon bond. The arylmoiety of mixed alkylaryl substituents R may be either carbocyclic orheterocyclic.

The symbol "Y" represents any substituent that meets the following tworequirements: First, the Y substituent, being a vinyl substituent of thealkene, must not highly activate group C═C as described above withrespect to substituents R₁ and R₂. Accordingly, Y may contain any of thedisclosed R₁ and R₂ substituents, except hydrogen, linked to C═C.Second, the Y substituent bears a functional group (hereinafter termed"Z") that is available for bonding to protein under mild conditions,such as acylation or aminidination, that preserve the biologicalactivity of the protein.

Functional group Z may be any activated functional group reactive withnucleophilic groups on a protein, protein fragment, antibody, antigenbinding fragment, amino acid polymer, plasma protein, peptide hormone,or mixtures thereof. By "activated functional group" is meant a Zsubstituent that is capable of reacting with and conjugating to anucleophilic substituent on a biologically active protein in aqueoussolution at a relatively rapid rate, under reaction conditions such astemperature and pH that do not denature or otherwise impair thebiological activity of the conjugated protein. Z is preferably aphenolic or imide ester, for covalent attachment to nucleophilicfunctional groups (or attachment sites) on amino acids or carbohydrateresidues of proteins or linking molecules that can in turn be bonded toprotein molecules. By "linking molecules" is meant bifunctionalconjugating reagents such as amino acid polymers, carbohydrates,dicarboxylic acids, diamines, and polyalcohols.

Alternatively, functional group Z may be a nucleophilic group suitablefor bonding with a derivatized protein or protein fragment containing anactivated functional group. Derivatized proteins reactive withnucleophilic functional groups Z include proteins that have been reactedwith cross-linking reagents such as homobifunctional imidoesters,homobifunctional N-hydroxysuccinimide (NHS) esters, andheterobifunctional cross-linkers having reactive functional groupsselected from NHS esters, maleimide, pyridyl disulfides, and activatedhalogens such as α-keto halides. Such cross-linking reagents arecommercially available from, e.g., Pierce Chemical Company, Rockford,IL. Accordingly, functional group Z may be an aldehyde, thiol, amine,carboxylate, alcohol, diazo, or groups reactive with a Michael-typeacceptor containing at least one α,β-unsaturated carbonyl such as amaleimide.

Generally, it is preferred that Y (as well as R₁ and R₂) be ashort-chain substituent since it is believed that unattached or cleavedradiohalogenated short-chain molecules are more rapidly removed by thekidneys. Thus, Y should preferably be a short-chain substituent havingan overall molecular length equivalent to no more than about 5, and mostpreferably no more than 3, straight-chain carbon atoms. Such ashort-chain Y may contain any of the suitably sized alkyl, heteroalkyl,aryl, heteroaryl, or mixed alkylaryl groups specified above for R₁ orR₂. Alternatively, for certain applications, Y may contain a longerspacer group between C═C and the functional group Z for proteinconjugation. Examples of such elongated spacer groups include, but arenot limited to, polypeptides and polysaccharides.

Representative radiohalogenated small molecules of this inventioncontaining Z groups are represented by formulas III and IV: ##STR4##wherein radiohalogen *X is substituted on C═C in either the cis, trans,or geminal (not shown) orientation with respect to substituent Y. Theinteger "n" is preferably 3 through 5. Suitable functional groups Z forthe above-stated purpose include phenolic esters (e.g., para-nitrophenylor tetrafluorophenyl ester), imide esters (e.g., succinimide ester),imidate esters, anhydrides, acylsuccinimides, diazo, hydrazines, alkylhalides (e.g., benzyl halide), and other groups that can be used toattach the molecule to a protein through a covalent bond. Preferred Zgroups include imide ester, alkyl imide esters, amido alkyl imideesters, imidate ester, alkyl imidate esters, amido alkyl imidate esters,and Michael-type acceptors such as maleimides.

Also provided are radiohalogenated small molecules of formulas I and IIwherein the Y substituent bears a precursor of the Z functional group.Suitable precursors include: carboxylic acid where Z is phenolic ester,imide ester, anhydride, acylsuccinimide, or maleimide; nitrile where Zis imidate ester; alcohol where Z is aldehyde; halide where Z isisothiocyanate, thiol, hydrazine, or amine; and amine where Z is diazoor maleimide.

While the spacer component (CH₂)_(n) is indicated in formula III, thespacer component in Y can be selected from the permissible R₁ and R₂substituents having equivalent lengths of up to about 12 but preferablyno more than about 5 straight-chain carbon atoms. Most preferably, nomore than three straight-chain carbon atoms separate functional group Zfrom the vinyl group, i.e., n=1, 2, or 3, in order to quickly clearbackground activity (any radiolabeled small molecule not attached to anantibody) for diagnostic imaging, and to minimize radiation dosage tonontargeted tissues.

In a most preferred embodiment, substituent Y has a fully substitutedcarbon atom directly bonded to C═C. This arrangement sterically hindersnucleophilic substitution on C═C, prevents migration of the double bond,and renders the spacer group relatively unavailable to enzymaticdegradation. A representative molecule of this type has the formula:##STR5## wherein *X, R₁, R₂, n, and Z are as stated above. The spacercomponent (CH₂)_(n) of substituent Y may alternatively be aryl,polynucleararyl, or heteroaryl.

Illustrative but nonlimiting examples of the subject radiohalogenatedsmall molecules include: ##STR6##

It is preferred that compounds having carboxyl groups as shown above bebonded to activated functional groups (e.g., succinimide ester ortetrafluorophenyl ester) to facilitate bonding of the radiohalogenatedsmall molecules to polypeptides, proteins, and protein fragments underconditions that preserve the biological activity of the proteincomponent of the conjugate. The above radiohalogenated small moleculesare also suitable, as shown, for direct reaction with polypeptides,proteins, and protein fragments that have been derivatized withbifunctional cross-linking reagents as described above.

Also provided are organometallic intermediate molecules of formulas VIor VII: ##STR7## wherein M is a metal-containing (metalated) groupsuitable for transmetalation or radiohalodemetalation, as describedbelow, and R₁, R₂, Y, and C═C are as defined above. M is suitably atrialkyl stannane such as Sn(n-Bu)₃ or SnMe₃, wherein Bu is butyl and Meis methyl, or is selected from among HgX, (X being Cl, Br, or I), HgOAc,(OAc being acetate), B(OH)₂, BX₂, BQ₂, (Q being hydride, alkyl, oralkoxy containing no more than about five, and preferably fewer, carbonatoms), Zr(cp)₂ Cl, (cp being cyclopentadienyl), and SiF₅ K₂.Illustrative but nonlimiting examples of the subject organometallicintermediate molecules include: ##STR8##

The compounds of formulas VI and VII can be prepared by a number ofmethods, including hydrometalating the corresponding alkynyl precursor,substituting an organometallic group for a halogen on the correspondingvinyl halide precursor, or transmetalating the corresponding vinyl metalor organometallic compound. Compounds suitable for hydrometalatingalkynyl precursors include: LiAlH₄, (alkyl)₂ AlH, tri-n-butyltinhydride,SnMe₃ H, Cl₃ SiH, and Zr(cp)₂ HCl.

Two general methods are provided for synthesizing the compounds offormulas iv, v, and vi. The first method employs a hydrometalationreaction to metalate an alkynyl derivative bearing the functional groupof Y or a precursor thereof. For example, the alkynyl derivative bearingthe functional group of Y corresponding to compound iv above would be:

    HC.tbd.CCH.sub.2 CH.sub.2 CO.sub.2 H                       (vii)

and a commercially available alkynyl derivative (Aldrich Chemical Co.,Milwaukee, WI) bearing a precursor to the functional group of Ycorresponding to compound iv above would be:

    HC.tbd.CCH.sub.2 CH.sub.2 CH.sub.2 OH                      (viii)

Furthermore, the alkynyl derivative bearing an activating functionalgroup Z of the Y substituent in vii would be: ##STR9## Any of thesecompounds may be hydrometalated; however, it is preferred tohydrometalate compound ix with (n-Bu)₃ SnH to produce the activated formof compound iv above.

The second synthesis method, termed a transmetalation reaction, employsthe site-specific conversion of one vinyl metal derivative into anothervinyl metal derivative. Vinyl metal derivatives can be transmetalatedwith one of the following groups: Sn(n-Bu)₃, SnMe₃, or other trialkylstannanes, HgX₂, Hg(OAc)₂, BX₃, BQ₃, Zr(cp)₂ Cl, or SiX₄, wherein X isCl, Br, or I, cp is cyclopentadienyl, OAc is acetate, and Q is hydride,alkyl, or alkoxy. In certain cases the transmetalated compound is tooreactive for effective radiohalogenation. These metalated compounds canbe converted to less reactive organometallic compounds which may be usedfor radiohalogenations. For example, ##STR10## can be converted to##STR11## with hydroxide ion, and ##STR12## can be converted to##STR13## with KF.

Suitable precursor molecules include: 4-pentynoic acid (Aldrich ChemicalCompany, Milwaukee, WI); 3,3-dimethyl 4-pentynoic acid (HelveticaChimica Acta 51: 1663-1678, 1968); and 4-ethynyl benzoic acid (J. Org.Chem. 43: 4491-4495, 1978).

Synthesis of vinylstannanes can be carried out via any one of thefollowing two distinctly different reactions. In the first reaction, thealkynyl derivative is reacted with tri-n-butyltin hydride at 60° C. orat room temperature in the presence of a radial initiator, such as AIBN(2,2-azobis(2-methylpropionitrile)). In the second reaction, a vinylhalide derivative, which can be prepared from the corresponding alkynylderivative, is reacted with n-butyl lithium at near -100° C. or withmagnesium at room temperature, followed by reaction of the vinyl metalwith a halide derivative of a trialkyltin reagent, preferablytri-n-butyltin chloride.

Synthesis of vinyl zirconium derivatives can be carried out by ahydrozirconation reaction of the alkynyl derivative using the zirconiumhydride, (cp)₂ ZrHCl, as described in Angew. Chem. Int. Ed. Engl. 15:333-340, 1976. Similarly, synthesis of vinyl boronic acid derivativescan be a hydroboration reaction of the alkynyl derivative, preferablyusing catechol borane, diborane, or BHCl₂, followed by hydrolysis.

Synthesis of the vinyl pentafluorosilicates can be carried out by anyone of the following two distinctly different reactions. In the firstreaction, the alkynyl derivative is hydrosilylated with trichlorosilane,followed by treatment of the intermediate vinyl trichlorosilylderivative with potassium fluoride. In the second reaction, a vinylhalide derivative is reacted with n-butyl lithium at near -100° C. orwith magnesium at room temperature, followed by reaction of the vinylmetal with silicon tetrachloride. The resulting vinyl trichlorosilylderivative is converted to the corresponding vinyl pentafluorosilicateas described above.

The preferred method for the synthesis of the vinyl mercurialderivatives is by transmetalation of either the above vinylstannanes orvinyl boronic esters. Reaction of a vinylstannane with Hg(OAc)₂ yieldsthe corresponding vinyl-HgOAc derivative in a site-specificsubstitution. J. Org. Chem. 22: 478, 1957. Likewise, reaction of a vinylboronic ester with Hg(OAc)₂ yields the corresponding vinyl HgOAc. J. Am.Chem. Soc. 94: 4371-4373, 1972. The vinyl-HgOAc can be further convertedto the vinyl Hg-X by reaction with halide ion (X).

Attaching the yet-to-be radiolabeled compounds to proteins will requirethe availability of a functional group Z, such as can be provided byconversion of a carboxylate precursor group into an ester containing agood leaving group, for example hydroxysuccinimide, or by conversion ofa cyano precursor into an imidate ester. Such conversions can beconsidered as activating the molecule towards reaction with acorresponding functional group, such as an amino group (e.g., lysineresidues), a thiol or hydroxy, (or, less preferably, a carboxylate), ona protein. Since metal hydrides, notably trialkyltin hydrides and boronhydrides, are reducing agents, when these reagents are used theactivated imide and imidate esters or other reduction sensitivefunctional groups Z can only be synthesized after the hydrometalationreaction. Making the activated imide and imidate esters or otherfunctional group Z prior to introducing the radiohalogen avoids lossesin radiochemical yields and the incorporation of radiochemicalimpurities that would otherwise result.

Conversion of the vinyl metalated derivatives from free carboxylic acidsor their stannyl esters to N-succinimidyl esters can be accomplishedprior to the radiohalogenation step, using dicyclohexylcarbodiimide(DCC) and N-hydroxysuccinimide (NHS) in anhydrous tetrahydrofuran (THF).However, synthesis of imidate esters from cyano compounds is madeproblematical by the acid liability of the vinyl-metal bond,particularly the vinyl-tin bond. Thus, cyano containing compounds may beconverted to the imidate prior to metalation or radiohalogenation andprior to formation of the imidate ester.

The vinyl metal derivatives are radiohalogenated via a halodemetalationreaction, preferably after the functional group Z is present.Radiohalogenation of the corresponding N-succinimidyl esters will yieldthe desired compounds via a site-specific demetalation reaction. Due tothe possibility of hydrolysis of the N-succinimidyl esters, thereactions should be carried out using conditions that will minimize thereaction time. For example, the reactants can be brought to roomtemperature in order to minimize the hydrolysis by shortening thedemetalation reaction time. Alternatively, reaction mixtures in whichthe hydrolysis is relatively slow can be used. For example, addition ofacetic acid to the reaction mixture significantly decreases the rate ofN-succinimidyl ester hydrolysis, advantageously eliminating the shorttime constraints routinely encountered with, e.g., the Bolton-Huntermethod.

The radiohalogenation reaction mixture should have a dilute sodiumthiosulfate solution added to it prior to any purification or workupprocedure. Separation of any remaining radiohalide can then beconveniently accomplished prior to or during purification of theradiolabeled protein via chromatographic separations.

The radiohalogenation reactions are preferably carried out in proticsolvents such as water, methanol, ethanol, or mixtures thereof. Thealcoholic solvents can be conveniently removed if needed prior toaddition of the radioactive compound to the protein solution, or viceversa. Alternatively, nonprotic solvents, e.g., carbon tetrachloride,can be used for radiohalogenation, since a biphasic system may provide aconvenient method of separating free radiohalide from the labeledcompounds.

The radiohalogenations can be monitored and purified by radio-HPLC, forexample on a reverse-phase high performance liquid chromatography column(C-18) eluted with a mixture of MeOH/1% HOAc in H₂ O.

It is contemplated that the enhanced stability and biodistribution ofsmall molecule radioiodinated proteins made in accordance with thisdisclosure will make such reagents remarkably suitable for bothdiagnosis and therapy.

Also provided are radiopharmaceutical kits for clinical use whichinclude a vial or set of vials containing any of compounds I throughVII, preferably bearing an activated functional group Z. Compounds VIand VII will typically be provided in combination with the appropriatebuffers and other reagents such that introduction of a radiohalogen willgive the desired radiohalogenated molecule. The radiohalogenated productcan then be attached to protein, such as a monoclonal antibody suppliedin a separate vial of the kit. The kit may also include one or morechromatographic columns or other suitable means for separating anyremaining precursors and impurities from the radiohalogenated proteinproduct.

Further provided is a method of stabilizing radiohalogens, via thesubject small molecules I and II, when attached to biologically activenon-protein molecules such as steroids and other hormonal molecules.

This invention is further illustrated by the following Examples.

EXAMPLE 1 Synthesis oftri-n-butylstannyl-5-(tri-n-butylstannyl)-4-pentenoate

A mixture of tri-n-butyltinhydride (2.0 equiv.) (Aldrich) and4-pentynoic acid (1.0 equiv.) (Aldrich) is warmed to 60° C. for sixhours. Kugelrohr distillation of the crude product givestri-n-butylstannyl 5-(tri-n-butylstannyl)-4-pentenoate acid.

EXAMPLE 2 Synthesis of 2,3,5,6-tetrafluorophenyl5-(tri-n-butylstannyl)-4-pentenoate

To a solution of tri-n-butylstannyl 5-(tri-n-butylstannyl)-4-pentenoateacid (1.0 equiv.) in anhydrous THF at room temperature is addeddicyclohexyl carbodiimide (1.2 equiv) (Aldrich) and2,3,5,6-tetrafluorophenol (1.2 equiv) (Aldrich). The resulting solutionis stirred overnight. The mixture is filtered, the filtrate isconcentrated, and the residue is chromatographed to provide2,3,5,6-tetrafluorophenyl 5-(tri-n-butylstannyl)-4-pentenoate.

EXAMPLE 3 Synthesis of tri-n-butylstannyl4-[2'-(tri-n-butylstannyl)ethenyl]benzoate

A mixture of 4-ethynylbenzoic acid (1.0 equiv.) andtri-n-butyltinhydride (1.0 equiv.) (Aldrich) is warmed to 60° C. for sixhours. Kugelrohr distillation of the crude product givestri-n-butylstannyl 4-[2'-(tri-n-butylstannyl)ethenyl]benzoate.

EXAMPLE 4 Synthesis of N-succinimidyl4-[2'-(tri-n-butylstannyl)ethenyl]benzoate

To a solution of tri-n-butylstannyl4-[2'-(tri-n-butylstannyl)ethenyl]-benzoate (1.0 equiv.) in anhydroustetrahydrofuran at room temperature is added dicyclohexyl carbodiimide(1.2 equiv.) (Aldrich) and N-hydroxysuccinimide (1.2 equiv.) (Aldrich).The resulting solution is stirred overnight. The mixture is filtered,the filtrate concentrated, and the residue is chromatographed to provideN-succinimidyl 4-[2'-(tri-n-butylstannyl)ethenyl]benzoate.

EXAMPLE 5 Radioiodination of N-succinimidyl4-[2-(tri-n-butylstannyl)ethenyl]benzoate

To a vial containing 10-50 μg (0.02-0.10 μmol) N-succinimidyl4-[2-(tri-n-butylstannyl)ethenyl]benzoate in 50 μL 5% aceticacid/methanol is added 10 μg (0.08 μmol) N-chlorosuccinimide in 10 μLmethanol. To this solution is added 10 μL Na¹²⁵ I solution (diluted inDelbecco's phosphate buffered saline; Gibco Labs) (100 μCi-2 mCi). After3-5 minutes, 10-20 μL of a 0.72 mg/ml aqueous solution of Na₂ S₂ O₅ isadded. The reaction mixture is concentrated by blowing a stream of N₂gas over the top of the solution until the volume is reduced to onlyaqueous. This material is used directly for protein labelingexperiments, as described below. Alternatively, radioiodination withiodine-131 is carried out in like manner.

EXAMPLE 6 Protein labeling with N-succinimidyl4-(2'-iodoethenyl)benzoate

The crude aqueous solution of N-succinimidyl 4-(2'-iodoethenyl)benzoateis transferred to a vial containing buffered protein solution (pH8.5-9), or vice versa. The conjugation is complete within 5 minutes atroom temperature. The labeled protein is purified from nonconjugatedradioactivity using either a gel permeation chromatography column or asmall pore filtration system (e.g., Centricon ultra centrifugation).

EXAMPLE 7 Radioiodination of 2,3,5,6-tetrafluorophenyl5-(tri-n-butylstannyl)-4-pentenoate

To a vial containing 10-50 μg (0.02-0.10 μmol) 2,3,5,6-tetrafluorophenyl5-(tri-n-butylstannyl)-4-pentenoate in 50 μL 5% acetic acid/methanol isadded 10 μg (0.08 μmol) N-chlorosuccinimide in 10 μL methanol. To thissolution is added 10 μL Na¹²⁵ I solution (diluted in Delbecco'sphosphate buffered saline; Gibco Labs) (100 μCi-2 mCi). After 3-5minutes, 10-20 μL of a 0.72 mg/mL aqueous solution of Na₂ S₂ O₅ isadded. The reaction mixture is concentrated by blowing a stream of N₂gas over the top of the solution until the volume is reduced to onlyaqueous. This material is used directly for protein labeling experimentsas described below. Radioiodination with iodine-131 is carried out inlike manner.

EXAMPLE 8 Protein labeling with 2,3,5,6-tetrafluorophenyl 5-[¹²⁵I]-iodo-4-pentenoate

The crude aqueous solution of 2,3,5,6-tetrafluorophenyl 5-[¹²⁵I]-iodo-4-pentenoate is transferred to a vial containing bufferedprotein solution (pH 8.5-10), or vice versa. The conjugation reaction iswarmed to 37° C. for 20 minutes. The labeled protein is purified fromnonconjugated radioactivity using either a gel permeation chromatographycolumn or a small pore filtration system (e.g., Centricon ultracentrifugation).

The radiohalogenated protein products can be used for radiodiagnosis andtherapy. For example, monoclonal antibodies or antigen binding fragmentsthat are specifically reactive with tumor cell associated antigens canbe radiohalogenated by this method and then used for imaging tumor celllocation in the body of a mammalian subject. An amount of theradiohalogenated antibody sufficient to achieve the imaging objective isintroduced, e.g., by intravenous injection, into the patient's body, andthereafter the body is scanned with a scintillation detector such as agamma camera. Such radiohalogenated antibodies can also be introducedinto the mammalian subject for the purpose of tumor radiotherapy.

Other proteins, protein fragments, modified proteins, and peptides thattend to concentrate in an organ or diseased tissue can likewise beradiohalogenated by this method and used to monitor departures fromhomeostasis and disease states. For example, radiohalogenated fibrinogencan be used to localize deep vein thrombosis by in vivo imaging.Disease-altered uptake of pituitary and other peptide hormones can bemonitored in a similar manner.

As a further example, antibodies radiohalogenated pursuant to thisdisclosure can be employed in in vitro radioimmunoassays.

All of the aforementioned radiohalogenated proteins are stablyradiolabeled because the radiohalogen is substituted onto a nonactivateddouble bond of the conjugate.

While the invention has been described in conjunction with preferredembodiments, one of ordinary skill after reading the foregoingspecification will be able to effect various changes, substitutions ofequivalents, and alterations to the subject matter set forth herein.Hence, the invention can be practiced in ways other than thosespecifically described herein. It is therefore intended that theprotection granted by Letters Patent hereon be limited only by theappended claims and equivalents thereof.

The embodiments of the invention in which an exclusive property orprivilege is claimed are defined as follows:
 1. A compound selected fromthe group consisting of tetrafluorophenyl ester of 5-*X-4-pentenoicacid, tetrafluorophenyl ester of 3,3-dimethyl-5-*X-4-pentenoic acid,tetrafluorophenyl ester of 4-[2'-*X-ethenyl]benzoic acid, N-succinimidylester of 5-*X-4-pentenoic acid, N-succinimidyl ester of3,3-dimethyl-5-*X-4-pentenoic acid, and N-succinimidyl ester of4-[2'-*X-ethenyl]benzoic acid, wherein *X is a radiohalogen.
 2. Acompound of claim 1 wherein *X is ¹²³ I, ¹²⁴ I, ¹²⁵ I, ¹³¹ I, ⁷⁵ Br, ⁷⁶Br, ⁷⁷ Br, ¹⁸ F or ²¹¹ At.